title: Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits.
abstract: Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10(1) to 10(2) bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.
authors.affiliation: AIT Austrian Institute of Technology, Molecular Medicine, A-1190 Vienna, Austria. herbert.wiesinger@ait.ac.at
authors.forename: Herbert
authors.initials: H
authors.lastname: Wiesinger-Mayr
authors.forename: Elena
authors.initials: E
authors.lastname: Jordana-Lluch
authors.forename: Elisa
authors.initials: E
authors.lastname: Martró
authors.forename: Silvia
authors.initials: S
authors.lastname: Schoenthaler
authors.forename: Christa
authors.initials: C
authors.lastname: Noehammer
mesh_terms: D001331:Automation / Q000379:methods*; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods*; D001774:Blood Chemical Analysis / Q000379:methods*; D017848:Blood-Borne Pathogens / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000097:blood* / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics; D006801:Humans; D046228:Microarray Analysis; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S